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Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

ABSTRACT: Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10??M ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96?hrs). Gas chromatography-mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12?hrs. Exposure for 48?hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.

SUBMITTER: Vernardis SI

PROVIDER: S-EPMC5292706 | biostudies-other | 2017 Feb

REPOSITORIES: biostudies-other

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Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

Vernardis Spyros I SI Terzoudis Konstantinos K Panoskaltsis Nicki N Mantalaris Athanasios A

Scientific reports 20170206

Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 μM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffec ...[more]

PMID: 28165055

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Project description:Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) are valuable for the understanding/treatment of the deadly heart diseases and their drug screening. However, the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging. Here, it is discovered surprisingly that Rock inhibitor (RI), used ubiquitously to improve the survival/yield of human iPSCs, induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 μM). This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 μM for 3D culture, the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95 % in ~7 days (compared to less than ~50 % in 14 days for the 10 μM RI condition). Furthermore, the outset beating time (OBT) of all resultant cardiac spheroids (CSs) is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol (GelMA) hydrogel, showing high homogeneity (in terms of the OBT) in functional maturity of the CSs. Moreover, the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs. These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases. Graphical abstract Rock inhibitor (RI) at 10 μM induces early gastrulation-like heterogeneous differentiation of human iPSCs and compromises their cardiac differentiation in 3D. Importantly, reducing RI to 1 μM retains human iPSCs in solid inner cell mass-like spheroids with high pluripotency for efficient and homogeneous cardiac differentiation in 3D. This discovery may greatly facilitate human iPSCs-based therapy and modeling of heart diseases.Image 1 Highlights • Rock inhibitor (RI) at the widely used 10 μM induces early gastrulation-like differentiation of human iPSCs (hiPSCs) in 3D.• Reducing RI to 1 μM retains the high pluripotency of hiPSCs in inner cell mass-like solid spheroids in 3D.• Reducing RI to 1 μM shortens the time needed for 3D cardiac differentiation of hiPSCs by ~1 week.• Reducing RI to 1 μM doubles the percentage of beating cardiac spheroids after 3D cardiac differentiation of hiPSCs.

Dataset Information

Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

Publications

Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

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